Project Summary/Abstract The microbiota within the human gastrointestinal (GI) tract is very diverse, and is unique to each individual at the genera/species level, but it is more generally conserved at the phyla level, with the predominant phyla being Bacteroidetes and Firmicutes, followed by Proteobacteria. The gut microbiota has been largely regarded as a resistance barrier towards enteric pathogens. However, the enteric pathogens enterohemorrhagic E. coli (EHEC) O157:H7 and Citrobacter rodentium (extensively used as a surrogate EHEC model for murine infections, given that EHEC does not infect mice), exploit cues and nutrients made available by members of the microbiota to regulate their virulence program. EHEC senses sugar sources such as fucose, and organic acids such as succinate to gauge the gastrointestinal (GI) environment to precisely regulate expression of its virulence repertoire. The relationship between EHEC and different members of the microbiota varies. Our studies using a representative member of each of the main phyla, Bacteroides thetatiotaomicron (Bacteroidetes), Enterococcus faecalis (Firmicutes) and commensal E. coli (Proteobacteria) suggest that EHEC virulence expression varies in response to these commensals, as well as to different combinations of them. Moreover, another important interaction between Bacteroides thetatiotaomicron and EHEC exist at the level of processing the type three secretion system, which promotes formation of attaching and effacing lesions (AE) on enterocytes. In this grant proposal we aim to address how different members of the microbiota impact enteric infections. It is notable that these studies will also be relevant to other enteric pathogens, such as Salmonella enterica and Clostridium difficile, among others, which share several of these pathogen-microbiota interaction strategies with EHEC. Hence the specific aims of this proposal are: Specific Aim 1. Investigate the impact of different commensals on EHEC and C. rodentium virulence in vitro and during murine infections. Specific Aim 2. Unravel how processing of the EHEC T3SS affects its function and EHEC virulence.